Phospholipids have a key role in contributing to the physicochemical properties of biological membranes. Because of the dynamic character of lipid-lipid and lipid-protein interactions in membranes and membrane mediated biological processes, characterization of the structure and dynamics of phospholipids in self-assembly and in response to addition of metabolites (e.g., other membrane components) is an important step toward understanding membrane biophysics as it relates to cell membrane function and signal transduction. The proposed research aims at characterization of the organization, structure and dynamic behavior of phospholipids in vesicle bilayers and micelles as membrane models using a series of newly designed double- labeled phospholipid analogues with strategically placed (site-directed) fluorescent reporter groups. The spectroscopic labels will be introduced (i) at each of the two fatty acyl chain-ends to characterize chain-chain interactions, and (ii) at the headgroup and each acyl chain, one at a time, to determine the distance / orientation between the polar headgroup and each one of the hydrophobic tails. These probes will be incorporated into vesicles and micelles of defined compositions and physicochemical parameters. Measurements of fluorescence resonance energy transfer (FRET) will be used to characterize the probes. The proposed hypothesis is that determination of the distance and orientation between the constituent fluorescent groups of phospholipid molecules in these synthetic analogues will provide a new technology to determine the structure and dynamics of phospholipid behavior in membranes. In contrast to the commonly used double- labeled fluorescent phospholipid analogues that are almost exclusively used for real-time spectroscopic assay of phospholipases by monitoring the loss of intramolecular fluorescence quenching, the spectroscopic probes here introduced are designed to be used as true spectroscopic rulers to determine the distance and orientation of the fluorescent groups in the phospholipid molecule by direct measurement of the FRET efficiency between the labels. Elucidation of phospholipid structure and dynamics in membranes and the way they interact with other membrane lipids and proteins will provide important information toward better understanding membrane behavior in health and disease.